The present invention relates to human flt3 receptor agonists. These flt3 receptor agonists retain one or more activities of native flt3 ligand and may also show improved hematopoietic cell-stimulating activity and/or an improved activity profile which may include reduction of undesirable biological activities associated with native flt3 ligand and/or have improved physical properties which may include increased solubility, stability and refold efficiency.
Colony stimulating factors which stimulate the differentiation and/or proliferation of bone marrow cells have generated much interest because of their therapeutic potential for restoring depressed levels of hematopoietic stem cell-derived cells. Colony stimulating factors in both human and murine systems have been identified and distinguished according to their activities. For example, granulocyte-CSF (G-CSF) and macrophage-CSF (M-CSF) stimulate the in vitro formation of neutrophilic granulocyte and macrophage colonies, respectively while GM-CSF and interleukin-3 (IL-3) have broader activities and stimulate the formation of both macrophage, neutrophilic and eosinophilic granulocyte colonies. Certain factors such as flt3 ligand are able to predominately affect stem cells.
Tyrosine kinase receptors are growth factor receptors that regulate the proliferation and differentiation of a number of cell. Certain tyrosine kinase receptors function within the hematopoietic system. Flt3 ligand (Rosnet et al., Oncogene, 6:1641-1650, 1991) and flk-2 (Matthews et al., Cell, 65:1143-1152, 1991) are forms of a tyrosine kinase receptor that is related to c-fms and c-kit receptors. The flk-2 and flt3 receptors are similar in amino acid sequence and vary at two amino acid residues in the extracellular domain and diverge in a 31 amino acid segment located near the C-terminus.
flt3 ligand is a hematopoietic growth factor which has the property of being able to regulate the growth and differentiation of hematopoietic progenitor and stem cells. Because of its ability to support the growth and proliferation of progenitor cells, flt3 receptor agonists have potential for therapeutic use in treating hematopoietic disorders such as aplastic anemia and myelodysplastic syndromes. Additionally, flt3 receptor agonists will be useful in restoring hematopoietic cells to normal amounts in those cases where the number of cells has been reduced due to diseases or to therapeutic treatments such as radiation and chemotherapy.
WO 94/28391 discloses the native flt3 ligand protein sequence and a cDNA sequence encoding the flt3 ligand, methods of expressing flt3 ligand in a host cell transfected with the cDNA and methods of treating patients with a hematopoietic disorder using flt3 ligand.
U.S. Pat. No. 5,554,512 is directed to human flt3 ligand as an isolated protein, DNA encoding the flt3 ligand, host cells transfected with cDNAs encoding flt3 ligand and methods for treating patients with flt3 ligand.
WO 94/26891 provides mammalian flt3 ligands, including an isolate that has an insertion of 29 amino acids, and fragments there of.
In evolution, rearrangements of DNA sequences serve an important role in generating a diversity of protein structure and function. Gene duplication and exon shuffling provide an important mechanism to rapidly generate diversity and thereby provide organisms with a competitive advantage, especially since the basal mutation rate is low (Doolittle, Protein Science 1:191-200, 1992).
The development of recombinant DNA methods has made it possible to study the effects of sequence transposition on protein folding, structure and function. The approach used in creating new sequences resembles that of naturally occurring pairs of proteins that are related by linear reorganization of their amino acid sequences (Cunningham, et al., Proc. Natl. Acad. Sci. U.S.A. 76:3218-3222, 1979; Teather and Erfle, J. Bacteriol. 172: 3837-3841, 1990; Schimming et al., Eur. J. Biochem. 204: 13-19, 1992; Yamiuchi and Minamikawa, FEBS Lett. 260:127-130, 1991: MacGregor et al., FEBS Lett. 378:263-266, 1996). The first in vitro application of this type of rearrangement to proteins was described by Goldenberg and Creighton (J. Mol. Biol. 165:407-413, 1983). A new N-terminus is selected at an internal site (breakpoint) of the original sequence, the new sequence having the same order of amino acids as the original from the breakpoint until it reaches an amino acid that is at or near the original C-terminus. At this point the new sequence is joined, either directly or through an additional portion of sequence (linker), to an amino acid that is at or near the original N-terminus, and the new sequence continues with the same sequence as the original until it reaches a point that is at or near the amino acid that was N-terminal to the breakpoint site of the original sequence, this residue forming the new C-terminus of the chain.
This approach has been applied to proteins which range in size from 58 to 462 amino acids (Goldenberg and Creighton, J. Mol. Biol. 165:407-413, 1983; Li and Coffino, Mol. Cell. Biol. 13:2377-2383, 1993). The proteins examined have represented a broad range of structural classes, including proteins that contain predominantly xcex1-helix (interleukin-4; Kreitman et al., Cytokine 7:311-318, 1995), xcex2-sheet (interleukin-1; Horlick et al., Protein Eng. 5:427-431, 1992), or mixtures of the two (yeast phosphoribosyl anthranilate isomerase; Luger et al., Science 243:206-210, 1989). Broad categories of protein function are represented in these sequence reorganization studies:
The results of these studies have been highly variable. In many cases substantially lower activity, solubility or thermodynamic stability were observed (E. coli dihydrofolate reductase, aspartate transcarbamoylase, phosphoribosyl anthranilate isomerase, glyceraldehyde-3-phosphate dehydrogenase, ornithine decarboxylase, omp A, yeast phosphoglycerate dehydrogenase). In other cases, the sequence rearranged protein appeared to have many nearly identical properties as its natural counterpart (basic pancreatic trypsin inhibitor, T4 lysozyme, ribonuclease T1, Bacillus xcex2-glucanase, interleukin-1xcex2, xcex1-spectrin SH3 domain, pepsinogen, interleukin-4). In exceptional cases, an unexpected improvement over some properties of the natural sequence was observed, e.g., the solubility and refolding rate for rearranged xcex1-spectrin SH3 domain sequences, and the receptor affinity and anti-tumor activity of transposed interleukin-4-Pseudomonas exotoxin fusion molecule (Kreitman et al., Proc. Natl. Acad. Sci. U.S.A. 91:6889-6893, 1994; Kreitman et al., Cancer Res. 55:3357-3363, 1995).
The primary motivation for these types of studies has been to study the role of short-range and long-range interactions in protein folding and stability. Sequence rearrangements of this type convert a subset of interactions that are long-range in the original sequence into short-range interactions in the new sequence, and vice versa. The fact that many of these sequence rearrangements are able to attain a conformation with at least some activity is persuasive evidence that protein folding occurs by multiple folding pathways (Viguera, et al., J. Mol. Biol. 247:670-681, 1995). In the case of the SH3 domain of xcex1-spectrin, choosing new termini at locations that corresponded to xcex2-hairpin turns resulted in proteins with slightly less stability, but which were nevertheless able to fold.
The positions of the internal breakpoints used in the studies cited here are found exclusively on the surface of proteins, and are distributed throughout the linear sequence without any obvious bias towards the ends or the middle (the variation in the relative distance from the original N-terminus to the breakpoint is ca. 10 to 80% of the total sequence length). The linkers connecting the original N- and C-termini in these studies have ranged from 0 to 9 residues. In one case (Yang and Schachman, Proc. Natl. Acad. Sci. U.S.A. 90:11980-11984, 1993), a portion of sequence has been deleted from the original C-terminal segment, and the connection made from the truncated C-terminus to the original N-terminus. Flexible hydrophilic residues such as Gly and Ser are frequently used in the linkers. Viguera, et al.(J. Mol. Biol. 247:670-681, 1995) compared joining the original N- and C-termini with 3-or 4-residue linkers; the 3-residue linker was less thermodynamically stable. Protasova et al. (Protein Eng. 7:1373-1377, 1994) used 3- or 5-residue linkers in connecting the original N-termini of E. coli dihydrofolate reductase; only the 3-residue linker produced protein in good yield.
SUMMARY OF THE INVENTION
The modified human flt3 receptor agonists of the present invention can be represented by the Formula:
X1xe2x88x92(L)axe2x88x92X2
wherein;
a is 0 or 1;
X1 is a peptide comprising an amino acid sequence corresponding to the sequence of residues n+1 through J;
X2 is a peptide comprising an amino acid sequence corresponding to the sequence of residues 1 through n;
n is an integer ranging from 1 to J-1; and
L is a linker.
In the formula above the constituent amino acids residues of human flt3 ligand are numbered sequentially 1 through J from the amino to the carboxyl terminus. A pair of adjacent amino acids within this protein may be numbered n and n+1 respectively where n is an integer ranging from 1 to J-1. The residue n+1 becomes the new N-terminus of the new flt3 receptor agonist and the residue n becomes the new C-terminus of the new flt3 receptor agonist.
The present invention relates to novel flt3 receptor agonists of the following formula:
wherein the N-terminus is joined to the C-terminus directly or through a linker capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids;
additionally said flt3 receptor agonist polypeptide can be immediately preceded by (methioninexe2x88x921), (alaninexe2x88x921) or (methionine xe2x88x922, alaninexe2x88x921).
A preferred embodiment is human flt3 receptor agonist polypeptide, comprising a modified flt3 ligand amino acid sequence of the Formula:
wherein the N-terminus is joined to the C-terminus directly or through a linker capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids;
additionally said flt3 receptor agonist polypeptide can be immediately preceded by (methioninexe2x88x921), (alaninexe2x88x921) or (methionine xe2x88x922, alaninexe2x88x921)
The more preferred breakpoints at which new C-terminus and N-terminus can be made are 36-37, 37-38, 38-39, 39-40, 40-41, 41-42, 42-43, 64-65, 65-66, 66-67, 86-87, 87-88, 88-89, 89-90, 90-91, 91-92, 92-93, 93-94, 95,-96, 96-97, 97-98, 99-100 and 100-101
The most preferred breakpoints at which new C-terminus and N-terminus can be made are; 39-40, 65-66, 89-90, 99-100 and 100-101.
The flt3 receptor agonists of the present invention may contain amino acid substitutions, deletions and/or insertions. It is also intended that the flt3 receptor agonists of the present invention may also have amino acid deletions at either/or both the N- and C-termini of the original protein and or deletions from the new N-and/or C-termini of the sequence rearranged proteins in the formulas shown above.
The flt3 receptor agonists of the present invention may contain amino acid substitutions, deletions and/or insertions.
A preferred embodiment of the present invention the linker (L) joining the N-terminus to the C-terminus is a polypeptide selected from the group consisting of:
GlyGlyGlySer SEQ ID NO:38;
GlyGlyGlySerGlyGlyGlySer SEQ ID NO:39;
GlyGlyGlySerGlyGlyGlySerGlyGlyGlySer SEQ ID NO:40;
SerGlyGlySerGlyGlySer SEQ ID NO:41;
GluPheGlyAsnMet SEQ ID NO:42;
GluPheGlyGlyAsnMet SEQ ID NO:43;
GluPheGlyGlyAsnGlyGlyAsnMet SEQ ID NO:44;
GlyGlySerAspMetAlaGly SEQ ID NO:45;
SerGlyGlyAsnGly SEQ ID NO:46;
SerGlyGlyAsnGlySerGlyGlyAsnGly SEQ ID NO:47;
SerGlyGlyAsnGlySerGlyGlyAsnGlySerGlyGlyAsnGly SEQ ID NO:48;
SerGlyGlySerGlySerGlyGlySerGly SEQ ID NO:49;
SerGlyGlySerGlySerGlyGlySerGlySerGlyGlySerGly SEQ ID NO:50;
GlyGlyGlySerGlyGly SEQ ID NO:51;
GlyGlyGlySerGlyGlyGly SEQ ID NO:52;
GlyGlyGlySerGlyGlyGlySerGlyGly SEQ ID NO:53;
GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGly SEQ ID NO:54;
GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGlyGly SEQ ID NO:55;
GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGly GlyGlySerGly SEQ ID NO:56;
GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGly GlyGlySerGlyGlyGlySerGlyGlyGlySerGly SEQ ID NO:148;
ProProProTrpSerProArgProLeuGlyAlaThrAlaProThrAlaGly GlnProProLeu SEQ ID NO:149;
ProProProTrpSerProArgProLeuGlyAlaThrAlaProThr SEQ ID NO:150; and
ValGluThrValPheHisArgValSerGlnAspGlyLeuLeuThrSer SEQ ID NO:151.
The present invention also encompasses recombinant human flt3 receptor agonists co-administered or sequentially with one or more additional colony stimulating factors (CSF) including, cytokines, lymphokines, interleukins, hematopoietic growth factors which include but are not limited to GM-CSF, G-CSF, c-mpl ligand (also known as TPO or MGDF), M-CSF, erythropoietin (FLT3), IL-1, IL-4, IL-2, IL-3, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, LIF, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor and stem cell factor (SCF) also known as steel factor or c-kit ligand (herein collectively referred to as xe2x80x9cfactorsxe2x80x9d). These co-administered mixtures may be characterized by having the usual activity of both of the peptides or the mixture may be further characterized by having a biological or physiological activity greater than simply the additive function of the presence of the flt3 receptor agonists or the second colony stimulating factor alone. The co-administration may also provide an enhanced effect on the activity or an activity different from that expected by the presence of the flt3 ligand or the second colony stimulating factor. The co-administration may also have an improved activity profile which may include reduction of undesirable biological activities associated with native human flt3 ligand. In addition to the list above, IL-3 variants taught in WO 94/12639 and WO 94/12638 fusion protein taught in WO 95/21197, and WO 95/21254 G-CSF receptor agonists disclosed in WO 97/12977, c-mpl receptor agonists disclosed in WO 97/12978, IL-3 receptor agonists disclosed in WO 97/12979 and multi-functional receptor agonists taught in WO 97/12985 can be co-administered with the polypeptides of the present invention. As used herein xe2x80x9cIL-3 variantsxe2x80x9d refer to IL-3 variants taught in WO 94/12639 and WO 94/12638. As used herein xe2x80x9cfusion proteinsxe2x80x9d refer to fusion protein taught in WO 95/21197, and WO 95/21254. As used herein xe2x80x9cG-CSF receptor agonistsxe2x80x9d refer to G-CSF receptor agonists disclosed in WO 97/12978. As used herein xe2x80x9cc-mpl receptor agonistsxe2x80x9d refer to c-mpl receptor agonists disclosed in WO 97/12978. As used herein xe2x80x9cIL-3 receptor agonistsxe2x80x9d refer to IL-3 receptor agonists disclosed in WO 97/12979. As used herein xe2x80x9cmulti-functional receptor agonistsxe2x80x9d refer to multi-functional receptor agonists taught in WO 97/12985.
In addition, it is envisioned that in vitro uses would include the ability to stimulate bone marrow and blood cell activation and growth before the expanded cells are infused into patients. Another intended use is for the production of dendritic cells both in vivo and ex vivo.
FIG. 1 schematically illustrates the sequence rearrangement of a protein. The N-terminus (N) and the C-terminus (C) of the native protein are joined through a linker, or joined directly. The protein is opened at a breakpoint creating a new N-terminus (new N) and a new C-terminus (new-C) resulting in a protein with a new linear amino acid sequence. A rearranged molecule may be synthesized de novo as linear molecule and not go through the steps of joining the original N-terminus and the C-terminus and opening of the protein at the breakpoint.
FIG. 2 shows a schematic of Method I, for creating new proteins in which the original N-terminus and C-terminus of the native protein are joined with a linker and different N-terminus and C-terminus of the protein are created. In the example shown the sequence rearrangement results in a new gene encoding a protein with a new N-terminus created at amino acid 97 of the original protein, the original C-terminus (a.a. 174) joined to the amino acid 11 (a.a. 1-10 are deleted) through a linker regionand a new C-terminus created at amino acid 96 of the original sequence.
FIG. 3 shows a schematic of Method II, for creating new proteins in which the original N-terminus and C-terminus of the native protein are joined without a linker and different N-terminus and C-terminus of the protein are created. In the example shown the sequence rearrangement results in a new gene encoding a protein with a new N-terminus created at amino acid 97 of the original protein, the original C-terminus (a.a. 174) joined to the original N-terminus and a new C-terminus created at amino acid 96 of the original sequence.
FIG. 4 shows a schematic of Method III, for creating new proteins in which the original N-terminus and C-terminus of the native protein are joined with a linker and different N-terminus and C-terminus of the protein are created. In the example shown the sequence rearrangement results in a new gene encoding a protein with a new N-terminus created at amino acid 97 of the original protein, the original C-terminus (a.a. 174) joined to amino acid 1 through a linker region and a new C-terminus created at amino acid 96 of the original sequence.
FIGS. 5a and 5b shows the DNA sequence encoding the 209 amino acid mature form of flt3 ligand from Lyman et al. (Oncogene 11:1165-1172, 1995).
FIG. 6 shows the DNA sequence encoding the 134 amino acid soluble form of flt3 ligand from Lyman et al. (Oncogene 11:1165-1172, 1995).
FIG. 7 shows the bioactivity of the flt3 receptor agonists pMON32320 and pMON32321 compared to recombinant native flt3 (Genzyme) and pMON30237 (1-134 form of the flt3 ligand expressed by mammalian cell (BHK) culture) in the MUTZ-2 cell proliferation assay. MT=mock transfection.